The immunological treatment of cancer aims to eradicate tumor cells based on their expression of antigens recognized by immune effector cells and molecules. One approach is to expand tumor-reactive T cells ex vivo and administer highly active effector cells capable of eradicating established tumors, referred to as adoptive cell therapy. The two essential challenges facing adoptive cell therapies are: (i) how to generate autologous T cells specific for cancer cells and (ii) how to sustain the efficacy of T cells after they have been infused in the recipient. The fundamental hypothesis underlying this hypothesis is that a genetic approach is potentially useful to enhance the anti-tumor activities of adoptively transferred T lymphocytes. The proposal is based on extensive experience with gene transfer in murine and human primary T lymphocytes and on two recent findings of ours on the targeting and co- stimulation of genetically modified primary T cells. In the first, we have shown that introduction of artificial receptor specific for prostate-specific membrane antigen (PSMA) and containing the cytoplasmic domain of the T cell receptor zeta chain (Pz-1) activates cytotoxicity and cytokine release by cancer patient T cells in the presence of cell-bound PSMA. In the second, we showed that an antigen-specific CD28-like co-stimulatory receptor is functional in human primary T cells, thus conferring protection against pro-apoptotic signals in the presence of the tumor antigen. This proposal explores molecular and cellular approaches to sustain and prolong the activity of tumor-specific cytotoxic T lymphocytes. The specific aims are: (1) To investigate whether tumor-specific cytotoxic T cells expressing a PSMA-specific CD28-like (P28-1) or 4-1BB-like (PBB-1) receptor show enhanced activity against PSMA* tumor cells. (2) To investigate whether PSMA-targeted CD8+T cells co-expressing zeta chain and co-stimulatory fusion receptors effectively eliminate PSMA- positive tumor cells in vitro and in vivo in mouse tumor models. (3) To investigate whether PSMA-targeted bone marrow-derived cells other than T lymphocytes that express Pz-1, P28-1 or PBB-1 play a role in tumor rejection in vivo. The latter studies investigate genetic approaches to recruit T cell help and natural killer cells to sustain the efficacy of tumor- specific cytotoxic T cells. The anticipated findings have important clinical implications for devising a therapeutic strategy against metastatic prostate cancer, for which there is no treatment, and for other cancers, such as breast and colon cancers, as PSMA is specifically expressed on the neovasculature of these tumors.